Guillermo Vasquez

Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.

The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2′-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2′-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5′-DNA/3′-MOE junction as well as the southern-biased 2′-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5′-MOE/3′-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.

Synthesis and biophysical properties of constrained D-altritol nucleic acids (cANA).

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the D-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA -1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure.

Synthesis and Incorporation of 2′-O-Methyl-Pseudouridine into Oligonucleotides

Abstract A short multigram synthesis of 2′-O-methylpseudouridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogens. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2′-O-methyluridine.

Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

2'-DMAOE RNA : EMERGING OLIGONUCLEOTIDES WITH PROMISING ANTISENSE PROPERTIES

Abstract Oligonucleotides with modifications at the carbohydrate 2′-position offer potential second-generation drug candidates1. ISIS 13312, a chimeric compound targeting CMV retinitis, has 2′-O-methoxyethyl2 (2′-MOE) modifications at the ends to offer enhanced binding affinity and nuclease resistance is an example of this trend. 2′-MOE modification offers high binding affinity and nuclease resistance presumably due to conformational constraints placed on the linkage by the oxygen-oxygen gauche effect3. On the other hand, 2′-O-aminopropyl modification (2′-AP) exhibits the highest nuclease resistance4, due to the presence of a cationic charge at the physiological pH. However, it lacks the binding affinity advantage of MOE due to the lack of oxygen-oxygen gauche effect. To optimize the antisense properties of both 2′-MOE and 2′-AP modifications, we have designed and constructed 2′-O-(aminooxyethyl) modification (2′-AOE)5 and 2′-O-(dimethylaminooxy ethyl) modification (2′-DMAOE) and synthesized oligomers hav...

An Efficient and Scalable Synthesis of Arabinosylguanine and 2′-Deoxy-2′-Fluoro-guanosine

Abstract An efficient conversion from commercially available 2, 6-diaminopurine-2′, 3′, 5′-tri-O-benzyl arabinoside to arabinosylguanine and its further transformation to 2′-deoxy-2′-fluoro-guanosine is outlined. This process has been used to produce more than one hundred grams of final product.

A NEW PROTECTING GROUP STRATEGY FOR AMINO GROUPS IN OLIGONUCLEOTIDE CHEMISTRY : CEOC GROUP

Abstract A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.